The long term objectives of this proposal are to characterize the role of defective interfering particles (DI) and other mutations in biological phenomena such as virus attenuation and persistence. Such mutations are undoubtedly important in chronic and degenerative diseases with a viral aetiology. To understand how these mutations work, we need to clarify the molecular mechanisms which control gene expression in these viruses. Vesicular stomatitis virus (VSV) and its DI represent a very useful model system for these detailed biochemical studies. Our specific aims are the following: 1) to characterize the viral RNA products synthesized by novel regulatory mutants of VSV; 2) to identify the precise mutational changes in the genome of these mutants; 3) to delineate the role of ribonucleoprotein template structure and/or conformation in regulating viral RNA synthesis; 4) to explore a novel electron microscopic approach for visualizing viral ribonucleoprotein templates actively synthesizing RNA; and 5) to establish a cell-free system capable of generating VSV DI de novo.